Mitochondria are organelles that generate virtually all of the energy for mammalian tissues. They are crucial to important cellular processes, such as programmed cell death, and abnormalities in their function are directly responsible for many diseases such as diabetes, heart disease, and stroke. Because the mitochondrial genome encodes only a few of the hundreds of proteins involved in their function, they must import most of their proteins across their double membranes. This process is complex and poorly understood in mammals, but defects in import can cause diseases such as methylmalonic acidemia. We have discovered a novel pathway whereby proteins are targeted and transported to the mitochondrial membranes. Furthermore, we have found a novel group of GTP-binding proteins in the mitochondrial membrane that regulates this process, and we are the only laboratory to have this data. In brief, precursor proteins destined for mitochondrial import are believed to be synthesized in the cytosol and imported by a receptor complex in the mitochondrial membrane (the post-translational import model). However, we have found that ribosomes specifically target and bind to the mitochondrial membrane. G-proteins, and the nature of the targeting sequence of the precursor protein, control this binding. The hypothesis behind this proposal is that import of proteins into mitochondria is a co-translational event, that is, ribosomes specifically target the mitochondrial surface and precursor proteins are imported as they are translated. This proposal has three specific aims: 1) Determine the mechanism of ribosome binding to mitochondria. Mutations in the transit peptide, competitive binding assays, and protease protection assays will identify the components of ribosome-mitochondrial binding. 2) Identify and analyze the ribosome receptor on the mitochondrial surface. Protease sensitivity of the receptor and its location will be determined. Competitive binding assays will determine the specificity of the receptor for the ribosome and its nascent chain. 3) Identify the mitochondrial membrane GTP- binding protein, G54. GTP hydrolysis controls ribosome binding to the mitochondrial membrane and one of these membrane G- proteins we recently discovered, termed G54, interacts with ribosomes. G54 will be purified and sequenced, and its nuclear gene and cDNA characterized. Identifying the components of this ribosome-mitochondrial interaction, and the function of these novel G-proteins, is crucial to understanding how mitochondria grow, divide, and repair themselves.